METABOLITES OF THE JANUS KINASE INHIBITOR (R)-3-(4-(7H-PYRROLO[2,3-d]PYRIMIDIN-4-YL)-1H-PYRAZOL-1-YL)-3-CYCLOPENTYLPROPANENITRILE

ABSTRACT

The present invention provides active metablites of 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile that modulate the activity of Janus kinases and are useful in the treatment of diseases related to activity of Janus kinases including, for example, immune-related diseases, skin disorders, myeloid proliferative disorders, cancer, and other diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Ser. No. 60/943,695, filedJun. 13, 2007, the disclosure of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides active metabolites of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilethat modulate the activity of Janus kinases and are useful in thetreatment of diseases related to activity of Janus kinases including,for example, immune-related diseases, skin disorders, myeloidproliferative disorders, cancer, and other diseases.

BACKGROUND OF THE INVENTION

Protein kinases (PKs) are a group of enzymes that regulate diverse,important biological processes including cell growth, survival anddifferentiation, organ formation and morphogenesis, neovascularization,tissue repair and regeneration, among others. Protein kinases exerttheir physiological functions through catalyzing the phosphorylation ofproteins (or substrates) and thereby modulating the cellular activitiesof the substrates in various biological contexts. In addition to thefunctions in normal tissues/organs, many protein kinases also play morespecialized roles in a host of human diseases including cancer. A subsetof protein kinases (also referred to as oncogenic protein kinases), whendysregulated, can cause tumor formation and growth, and furthercontribute to tumor maintenance and progression (Blume-Jensen P et al,Nature 2001, 411(6835):355-365). Thus far, oncogenic protein kinasesrepresent one of the largest and most attractive groups of proteintargets for cancer intervention and drug development.

The Janus Kinase (JAK) family plays a role in the cytokine-dependentregulation of proliferation and function of cells involved in immuneresponse. Currently, there are four known mammalian JAK family members:JAK1 (also known as Janus kinase-1), JAK2 (also known as Januskinase-2), JAK3 (also known as Janus kinase, leukocyte; JAKL; L-JAK andJanus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). TheJAK proteins range in size from 120 to 140 kDa and comprise sevenconserved JAK homology (JH) domains; one of these is a functionalcatalytic kinase domain, and another is a pseudokinase domainpotentially serving a regulatory function and/or serving as a dockingsite for STATs (Scott, Godshall et al. 2002, supra).

Blocking signal transduction at the level of the JAK kinases holdspromise for developing treatments for human cancers. Inhibition of theJAK kinases is also envisioned to have therapeutic benefits in patientssuffering from skin immune disorders such as psoriasis, and skinsensitization. Accordingly, inhibitors of Janus kinases or relatedkinases are widely sought and several publications report effectiveclasses of compounds. For example, certain JAK inhibitors, including(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrileshown below, are reported in U.S. Ser. No. 11/637,545, filed Dec. 12,2006.

Thus, new or improved agents which inhibit kinases such as Janus kinasesare continually needed for developing new and more effectivepharmaceuticals to treat cancer and other diseases. The metabolites,compositions and methods described herein are directed toward theseneeds and other ends.

SUMMARY OF THE INVENTION

The present invention provides a compound selected from:

-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;    and-   3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxocyclopentyl)propanenitrile;

or pharmaceutically acceptable salt thereof.

The present invention further provides one or more of the abovecompounds, or pharmaceutically acceptable salts thereof, insubstantially isolated form.

The present invention further provides compositions comprising acompound of the invention, or pharmaceutically acceptable salt thereof,and at least one pharmaceutically acceptable carrier.

The present invention further provides methods of modulating an activityof JAK comprising contacting JAK with a compound of the presentinvention, or pharmaceutically acceptable salt thereof.

The present invention further provides methods of treating a disease ina patient, comprising administering to the patient a therapeuticallyeffective amount of a compound of the invention, or pharmaceuticallyacceptable salt thereof.

DETAILED DESCRIPTION

The present invention provides, inter alia, compounds that are activemetabolites of the JAK inhibitor(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile.These metabolites modulate the activity of one or more JAKs and areuseful, for example, in the treatment of diseases associated with JAKexpression or activity. The metabolites of the invention are indicatedin Table 1 below. Structures are intended to encompass all possiblestereoisomers.

TABLE 1 Reference Name Structure Metabolite 1 3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)- 1H-pyrazol-1-yl)-3-(3- hydroxycyclopentyl)propanenitrile

Metabolite 2 3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2- hydroxycyclopentyl) propanenitrile

Metabolite 3 3-(4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3- oxocyclopentyl) propanenitrile

The metabolites of the invention were isolated from rat or dog urinesamples collected from pharmacokinetic and toxicokinetic studies of theJAK inhibitor(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile(Compound 1). As shown in Table 2 and detailed in Example A, themetabolites are active and potent JAK inhibitors, and have advantageousproperties related to significantly higher free fractions and highermetabolic stability in human microsomes compared with Compound 1. Thisdata suggests the present metabolites may desirably have a longerelimination half-life in humans than does Compound 1.

In some embodiments, the metabolites of the invention are substantiallyisolated. By “substantially isolated” is meant that the compound is atleast partially or substantially separated from the environment in whichit was formed or detected. Partial separation can include, for example,a composition enriched in the compound of the invention. Substantialseparation can include compositions containing at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 97%, or at least about 99% byweight of the metabolite.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in its entirety.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The metabolites are asymmetric (e.g., having one or more stereocenters).All stereoisomers, such as enantiomers and diastereomers, are intendedunless otherwise indicated. Methods on how to prepare optically activeforms from optically active starting materials are known in the art,such as by resolution of racemic mixtures or by stereoselectivesynthesis.

Compounds of the invention also include all isotopes of atoms occurringin the metabolites. Isotopes include those atoms having the same atomicnumber but different mass numbers. For example, isotopes of hydrogeninclude tritium and deuterium.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

Synthesis

Compounds of the invention, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., Wiley & Sons,Inc., New York (1999), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), ormass spectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Compounds of the invention can be prepared according to numerouspreparatory routes known in the literature. Example synthetic methodsfor preparing compounds of the invention are provided in the Schemesbelow.

As shown in Scheme 1, synthesis of the diastereomeric mixture of the cisalcohols I starts with cyclopentene carboxylic acid 1. Cyclopentenecarboxylic acid 1 is bromolactonized following a procedure describedearlier (Hodgson, David M.; Witherington, Jason; Moloney, Brian A.,Journal of the Chemical Society, Perkin Transactions 1: Organic andBio-Organic Chemistry, 1994, 23, 3950) to give the correspondingbromolactone 2. The bromolactone 2 is debrominated with the use of adehalogenating agent, such as (Me₃Si)₃SiH to give 3. The lactone 3 isreduced to the corresponding hemiketal with the use of a reducing agent,such as DIBAL-H; the hemiketal formed is treated directly with the ylid3a to give the crotonitrile derivative 4. The nitrile 4 then reacts withthe pyrazole 5 in the presence of a base such as DBU to give 6 as amixture of diastereomers, which is converted to the alcohols I afterremoval of the SEM group. The individual stereoisomers of this mixture(I) can be separated by chiral chromatography to give theenantiomerically pure alcohols (4 total stereoisomers).

As shown in Scheme 2, the synthesis of the trans alcohols II starts withthe diastereomeric mixture of alcohols 6. The diastereomeric mixture ofalcohols 6 is treated with benzoic acid under the Mitsunobu conditionsto give a mixture of the trans benzoates 7 with complete inversion. Themixture of the benzoates 7 is hydrolyzed by treatment with a base suchas LiOH to give a mixture of the trans alcohols 8. The SEM group withinthe alcohols 8 is then removed to give the diastereomeric mixture of thetrans alcohols II, which is separated by chiral chromatography to giveindividual stereoisomers (4 total stereoisomers).

The synthesis of the ketones III is described in Scheme 3. A mixture ofthe cis alcohols 6 can be oxidized under Swern conditions to give thecorresponding mixture of ketones 9. The SEM group within the ketones 9is removed to give a mixture of the ketone III, which can be separatedby chiral chromatography to give the individual stereoisomers (4 totalstereoisomers).

Methods

Compounds of the invention can modulate activity of one or more Januskinases (JAKs). The term “modulate” is meant to refer to an ability toincrease or decrease the activity of one or more members of the JAKfamily of kinases. Accordingly, compounds of the invention can be usedin methods of modulating a JAK by contacting the JAK with any one ormore of the compounds or compositions described herein. In someembodiments, compounds of the present invention can act as inhibitors ofone or more JAKs. In some embodiments, compounds of the presentinvention can act to stimulate the activity of one or more JAKs. Infurther embodiments, the compounds of the invention can be used tomodulate activity of a JAK in an individual in need of modulation of thereceptor by administering a modulating amount of a compound of theinvention.

JAKs to which the present compounds bind and/or modulate include anymember of the JAK family. In some embodiments, the JAK is JAK1, JAK2,JAK3 or TYK2. In some embodiments, the JAK is JAK1 or JAK2. In someembodiments, the JAK is JAK2. In some embodiments, the JAK is JAK3.

The compounds of the invention can be selective. By “selective” is meantthat the compound binds to or inhibits a JAK with greater affinity orpotency, respectively, compared to at least one other JAK. In someembodiments, the compounds of the invention are selective inhibitors ofJAK1 or JAK2 over JAK3 and/or TYK2. In some embodiments, the compoundsof the invention are selective inhibitors of JAK2 (e.g., over JAK1, JAK3and TYK2). Without wishing to be bound by theory, because inhibitors ofJAK3 can lead to immunosuppressive effects, a compound which isselective for JAK2 over JAK3 and which is useful in the treatment ofcancer (such as multiple myeloma, for example) can offer the additionaladvantage of having fewer immunosuppressive side effects. Selectivitycan be at least about 5-fold, 10-fold, at least about 20-fold, at leastabout 50-fold, at least about 100-fold, at least about 200-fold, atleast about 500-fold or at least about 1000-fold. Selectivity can bemeasured by methods routine in the art. In some embodiments, selectivitycan be tested at the Km of each enzyme. In some embodiments, selectivityof compounds of the invention for JAK2 over JAK3 can be determined bythe cellular ATP concentration.

Another aspect of the present invention pertains to methods of treatinga JAK-associated disease or disorder in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a compound of the presentinvention or a pharmaceutical composition thereof. A JAK-associateddisease can include any disease, disorder or condition that is directlyor indirectly linked to expression or activity of the JAK, includingoverexpression and/or abnormal activity levels. A JAK-associated diseasecan also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating JAK activity.

Examples of JAK-associated diseases include diseases involving theimmune system including, for example, organ transplant rejection (e.g.,allograft rejection and graft versus host disease).

Further examples of JAK-associated diseases include autoimmune diseasessuch as multiple sclerosis, rheumatoid arthritis, juvenile arthritis,type I diabetes, lupus, psoriasis, inflammatory bowel disease,ulcerative colitis, Crohn's disease, myasthenia gravis, immunoglobulinnephropathies, autoimmune thyroid disorders, and the like. In someembodiments, the autoimmune disease is an autoimmune bullous skindisorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditionssuch as asthma, food allergies, atopic dermatitis and rhinitis. Furtherexamples of JAK-associated diseases include viral diseases such asEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicella-Zoster Virus (VZV) and Human Papilloma Virus (HPV).

Further examples of JAK-associated diseases or conditions include skindisorders such as psoriasis (for example, psoriasis vulgaris), atopicdermatitis, skin rash, skin irritation, skin sensitization (e.g.,contact dermatitis or allergic contact dermatitis). For example, certainsubstances including some pharmaceuticals when topically applied cancause skin sensitization. In some embodiments, co-administration orsequential administration of at least one JAK inhibitor of the inventiontogether with the agent causing unwanted sensitization can be helpful intreating such unwanted sensitization or dermatitis. In some embodiments,the skin disorder is treated by topical administration of at least oneJAK inhibitor of the invention.

In further embodiments, the JAK-associated disease is cancer includingthose characterized by solid tumors (e.g., prostate cancer, renalcancer, hepatic cancer, pancreatic cancer, gastric cancer, breastcancer, lung cancer, cancers of the head and neck, thyroid cancer,glioblastoma, Kaposi's sarcoma, Castleman's disease, melanoma etc.),hematological cancers (e.g., lymphoma, leukemia such as acutelymphoblastic leukemia, acute myelogenous leukemia (AML) or multiplemyeloma), and skin cancer such as cutaneous T-cell lymphoma (CTCL) andcutaneous B-cell lymphoma. Example cutaneous T-cell lymphomas includeSezary syndrome and mycosis fungoides.

JAK-associated diseases can further include those characterized byexpression of a mutant JAK2 such as those having at least one mutationin the pseudo-kinase domain (e.g., JAK2V617F).

JAK-associated diseases can further include myeloproliferative disorders(MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET),myeloid metaplasia with myelofibrosis (MMM), chronic myelogenousleukemia (CML), chronic myelomonocytic leukemia (CMML),hypereosinophilic syndrome (HES), systemic mast cell disease (SMCD), andthe like.

Further JAK-associated diseases include inflammation and inflammatorydiseases. Example inflammatory diseases include inflammatory diseases ofthe eye (e.g., iritis, uveitis, scleritis, conjunctivitis, or relateddisease), inflammatory diseases of the respiratory tract (e.g., theupper respiratory tract including the nose and sinuses such as rhinitisor sinusitis or the lower respiratory tract including bronchitis,chronic obstructive pulmonary disease, and the like), inflammatorymyopathy such as myocarditis, and other inflammatory diseases. Otherinflammatory diseases treatable by the compounds of the inventioninclude systemic inflammatory response syndrome (SIRS) and septic shock.

The JAK inhibitors described herein can further be used to treatischemia reperfusion injuries or a disease or condition related to aninflammatory ischemic event such as stroke or cardiac arrest. The JAKinhibitors described herein can further be used to treat anorexia,cachexia, or fatigue such as that resulting from or associated withcancer. The JAK inhibitors described herein can further be used to treatrestenosis, sclerodermitis, or fibrosis. The JAK inhibitors describedherein can further be used to treat conditions associated with hypoxiaor astrogliosis such as, for example, diabetic retinopathy, cancer, orneurodegeneration. See, e.g., Dudley, A. C. et al. Biochem. J. 2005,390(Pt 2):427-36 and Sriram, K. et al. J. Biol. Chem. 2004,279(19):19936-47. Epub 2004 Mar. 2.

The JAK inhibitors described herein can further be used to treat goutand increased prostate size due to, e.g., benign prostatic hypertrophyor benign prostatic hyperplasia.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a JAK with a compound of the invention includesthe administration of a compound of the present invention to anindividual or patient, such as a human, having a JAK, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the JAK.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder; and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinaseinhibitors such as, for example, those described in WO 2006/056399, orother agents can be used in combination with the compounds of thepresent invention for treatment of JAK-associated diseases, disorders orconditions. The one or more additional pharmaceutical agents can beadministered to a patient simultaneously or sequentially.

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, EP2005/009967, EP2005/010408, and U.S.Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

In some embodiments, one or more of the metabolites of the invention canbe used in combination with one or more other kinase inhibitorsincluding imatinib, particularly for treating patients resistant toimatinib or other kinase inhibitors.

In some embodiments, one or more JAK inhibitors of the invention can beused in combination with a chemotherapeutic in the treatment of cancer,such as multiple myeloma, and may improve the treatment response ascompared to the response to the chemotherapeutic agent alone, withoutexacerbation of its toxic effects. Examples of additional pharmaceuticalagents used in the treatment of multiple myeloma, for example, caninclude, without limitation, melphalan, melphalan plus prednisone [MP],doxorubicin, dexamethasone, and Velcade (bortezomib). Further additionalagents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,RAF and FAK kinase inhibitors. Additive or synergistic effects aredesirable outcomes of combining a JAK inhibitor of the present inventionwith an additional agent. Furthermore, resistance of multiple myelomacells to agents such as dexamethasone may be reversible upon treatmentwith a JAK inhibitor of the present invention. The agents can becombined with the present compounds in a single or continuous dosageform, or the agents can be administered simultaneously or sequentiallyas separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with at least one JAK inhibitorwhere the dexamethasone is administered intermittently as opposed tocontinuously.

In some further embodiments, combinations of one or more JAK inhibitorsof the invention with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the compounds of the inventionabove in combination with one or more pharmaceutically acceptablecarriers (excipients). In making the compositions of the invention, theactive ingredient is typically mixed with an excipient, diluted by anexcipient or enclosed within such a carrier in the form of, for example,a capsule, sachet, paper, or other container. When the excipient servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, carrier or medium for the active ingredient. Thus,the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active compound,soft and hard gelatin capsules, suppositories, sterile injectablesolutions, and sterile packaged powders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, for example see InternationalPatent Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

The active compound can be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face masks tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present invention canvary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound, thehealth and condition of the patient, and the judgment of the prescribingphysician. The proportion or concentration of a compound of theinvention in a pharmaceutical composition can vary depending upon anumber of factors including dosage, chemical characteristics (e.g.,hydrophobicity), and the route of administration. For example, thecompounds of the invention can be provided in an aqueous physiologicalbuffer solution containing about 0.1 to about 10% w/v of the compoundfor parenteral administration. Some typical dose ranges are from about 1μg/kg to about 1 g/kg of body weight per day. In some embodiments, thedose range is from about 0.01 mg/kg to about 100 mg/kg of body weightper day. The dosage is likely to depend on such variables as the typeand extent of progression of the disease or disorder, the overall healthstatus of the particular patient, the relative biological efficacy ofthe compound selected, formulation of the excipient, and its route ofadministration. Effective doses can be extrapolated from dose-responsecurves derived from in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled compounds ofthe invention (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in imaging techniques but also in assays, both in vitroand in vivo, for localizing and quantitating JAK in tissue samples,including human, and for identifying JAK ligands by inhibition bindingof a labeled compound. Accordingly, the present invention includes JAKassays that contain such labeled compounds.

The present invention further includes isotopically-labeled compounds ofthe invention. An “isotopically” or “radio-labeled” compound is acompound of the invention where one or more atoms are replaced orsubstituted by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number typically found in nature (i.e.,naturally occurring). Suitable radionuclides that may be incorporated incompounds of the present invention include but are not limited to ²H(also written as D for deuterium), ³H (also written as T for tritium),¹¹C, ¹³C, ¹⁴N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br,⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. The radionuclide that is incorporatedin the instant radio-labeled compounds will depend on the specificapplication of that radio-labeled compound. For example, for in vitrometalloprotease labeling and competition assays, compounds thatincorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be mostuseful. For radio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I,⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments the radionuclide is selected from the group consisting of³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br.

The present invention can further include synthetic methods forincorporating radio-isotopes into compounds of the invention. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and a person of ordinary skill in the art will readilyrecognize the methods applicable for the compounds of invention.

A labeled compound of the invention can be used in a screening assay toidentify/evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a JAK by monitoring its concentrationvariation when contacting with the JAK, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a JAK (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the JAKdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of JAK-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of the invention. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of noncriticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES Example 13-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Step 1. 6-bromo-2-oxabicyclo[2.2. 1]heptan-3-one

Bromotrimethysilane (3.1 mL, 0.023 mol) was added dropwise to a solutionof dimethyl sulfoxide (1.6 mL, 0.023 mol) in chloroform (38.0 mL) in around-bottom flask at 0° C. The resulting mixture was stirred at 0° C.for 2 hours. To the reaction mixture was added dropwise a solution ofcyclopent-3-ene-1-carboxylic acid (2.00 g, 0.0178 mol) in chloroform (12mL) over a period of 15 minutes and the reaction mixture was stirred at0° C. for 10 minutes. N,N-diisopropylethylamine (4.0 mL, 0.023 mol) wasthen added and the resulting mixture was stirred at 0° C. After 10minutes, the mixture was heated to reflux for 16 hours. The reactionmixture was diluted with chloroform, washed with water, brine, dried(MgSO₄), and stripped in vacuo. The residue was purified bychromatography on silica gel using 30% EtOAc/hexanes as eluent to givethe product. ¹H NMR (400 MHz, CDCl₃): δ 4.88 (brs, 1H), 4.34 (m, 1H),2.90 (m, 1H), 2.66 (m, 1H), 2.31 (m, 1H), 1.93 (m, 1H), 1.83 (m, 1H).

Step 2. 2-oxabicyclo[2.2.1]heptan-3-one

Tris(trimethylsilyl)silane (4.7 mL, 15 mmol) was added dropwise to asolution of 6-bromo-2-oxabicyclo[2.2.1]heptan-3-one (1.96 g, 10.3 mmol)and 2,2′-azo-bis-isobutyronitrile (0.2 g, 1 mmol) in toluene (100 mL) ina round-bottom flask and the resulting mixture was stirred at 80° C. for5 hours. The reaction mixture was concentrated by rotoary evaporationand the residue was diluted with ethyl acetate, washed with saturatedNH₄Cl, dried (MgSO₄), and stripped in vacuo. The residue was purified bychromatography on silica gel using 100% hexanes, grading to 25%EtOAc/hexanes then 33% EtOAc/hexanes as eluents to give the product. ¹HNMR (300 MHz, CDCl₃): δ 4.93 (m, 1H), 2.91 (m, 1H), 2.19 (m, 1H),1.60-1.99 (m, 5H).

Step 3. (2E)- and (2Z)-3-[(1S,3R)-3-hydroxycyclopentyl]acrylonitrile and(2E)- and (2Z)-3-[(1R,3S)-3-hydroxycyclopentyl]acrylonitrile

1.00 M of diisobutylaluminum hydride in toluene (8.0 mL) was addeddropwise to a solution of 2-oxabicyclo[2.2.1]heptan-3-one (600 mg, 5mmol) in methylene chloride (20 mL) in a round-bottom flask at −78° C.The resulting mixture was stirred at −78° C. for 45 minutes. Thereaction mixture was treated with saturated Rochelle's salt solution.After stirring for 15 minutes, the reaction mixture was extracted withethyl acetate and the combined organic extracts were washed with water,saturated NaCl, dried (MgSO₄), and stripped in vacuo. The crude productwas used in the next reaction without further purification.

A solution of the crude 2-oxabicyclo[2.2.1]heptan-3-ol (400 mg, 4 mmol)and (triphenylphosphoranylidene)acetonitrile (1.2 g, 3.8 mmol) intoluene (12 mL) in a round-bottom flask was heated at 80° C. for 2hours. The reaction mixture was then purified by chromatography onsilica gel using 40% EtOAc/hexanes to give the racemic products. ¹H NMR(400 MHz, CDCl₃): δ 6.78 (dd, 1H), 5.30 (d, 1H), 5.20 (m, 1H), 2.67 (m,1H), 2.20 (m, 1H), 1.40-1.90 (m, 6H).

Step 4.3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

1,8-Diazabicyclo[5.4.0]undec-7-ene (0.54 mL, 3.6 mmol) was added to asolution of a mixture of (2E)- and(2Z)-3-[(1S,3R)-3-hydroxycyclopentyl]acrylonitrile and (2E)- and(2Z)-3-[(1R,3S)-3-hydroxycyclopentyl]acrylonitrile (0.250 g, 1.82 mmol)and4-(1H-pyrazol-4-yl)-7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidine(0.57 g, 1.8 mmol) in acetonitrile (5 mL) in a round-bottom flask. Theresulting mixture was stirred at 25° C. for 2 days at which time LCMSanalysis showed ˜80% of the starting materials had been consumed. Thereaction mixture was purified by chromatography on silica gel using 1:1EtOAc/hexanes to give the product. ¹H NMR (400 MHz, CDCl₃): δ 8.90 (d,1H), 8.39 (m, 2H), 7.46 (m, 1H), 6.86 (m 1H), 5.73 (s, 2H), 4.52 (m,2H), 3.59 (m, 2H), 3.2 (m, 1H), 3.02 (m, 1H), 2.78 (m, 1H), 2.3 (m, 1H),1.30-1.90 (m, 6H), 0.99 (m, 2H), 0.08 (s, 9H). LC/MS: 453 (M+H)⁺.

Step 5.3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Lithium tetrafluoroborate (0.176 g, 1.88 mmol) was added to a solutionof3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(85.0 mg, 0.188 mmol) in acetonitrile (1.5 mL) and water (0.135 mL) in avial. The resulting mixture was heated at 85° C. for 26 hours. After thereaction mixture was allowed to cool to 25° C., ethylenediamine (63 μL,0.94 mmol) was added and the resulting mixture was stirred at 25° C. for3 hours. The reaction mixture was purified by prep. LC to give theproduct as the trifluoroacetic acid salt. This was dissolved in methanoland Amberlyst 26 was added. The resulting mixture was stirred for 10minutes, filtered, and concentrated. The residue was purified by chiralchromatography to give 4 major peaks and 4 minor peaks. (Column:ChiralPak IA, 4.6×250 mm, 5 micron particle. Mobile phase: 30% Ethanolin hexanes. Flow Rate: 0.8 ml/min-analytical; Column: ChiralPak IA,20×250 mm, 5 micron particle. Mobile Phase: 30% Ethanol in hexanes. Flowrate: 12 ml/min-preparative) The minor peaks were attributed to thetrifluoroacetate esters which are very mobile and are cleaved onstanding in methanol to the corresponding alcohols.

Major Peak 1 [Retention time: 18.56 minutes]: ¹H NMR (400 MHz, CD₃OD): δ8.66 (brs, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 7.51 (m, 1H), 6.97 (m, 1H),4.57 (m, 1H), 4.20 (m, 1H), 3.16 (m, 2H), 2.65 (m, 1H), 1.64-2.00 (m,5H), 1.28 (m, 1H). LC/MS: 323 (M+H)⁺.

Major Peak 2 [Retention time: 25.88 minutes]: ¹H NMR (400 MHz, CD₃OD): δ8.66 (brs, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 7.50 (m, 1H), 6.96 (m, 1H),4.60 (m, 1H), 4.30 (m, 1H), 3.18 (m, 2H), 2.61 (m, 1H), 2.23 (m, 1H),1.40-1.80 (m, 5H). LC/MS: 323 (M+H)⁺.

Major Peak 3 [Retention time: 39.84 minutes]: ¹H NMR (400 MHz, CD₃OD): δ8.66 (brs, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 7.50 (m, 1H), 6.96 (m, 1H),4.60 (m, 1H), 4.30 (m, 1H), 3.18 (m, 2H), 2.61 (m, 1H), 2.23 (m, 1H),1.40-1.80 (m, 5H). LC/MS: 323 (M+H)⁺.

Major Peak 4 [Retention time: 51.48 minutes]: ¹H NMR (400 MHz, CD₃OD): δ8.66 (brs, 1H), 8.64 (s, 1H), 8.38 (s, 1H), 7.51 (m, 1H), 6.97 (m, 1H),4.57 (m, 1H), 4.20 (m, 1H), 3.16 (m, 2H), 2.65 (m, 1H), 1.64-2.00 (m,5H), 1.28 (m, 1H). LC/MS: 323 (M+H)⁺.

Example 23-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt and3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt

Step 1:(1S,3S)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentylbenzoate and(1R,3R)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentylbenzoate

Diisopropyl azodicarboxylate (0.38 mL, 1.9 mmol) was added to a solutionof3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(0.51 g, 1.9 mmol) in tetrahydrofuran (5.3 mL) in a round-bottom flaskat 0° C. The resulting mixture was stirred for 10 minutes and benzoicacid (0.24 g, 1.9 mmol) was added. The reaction mixture was stirred at0° C. for 2 hours at which time TLC analysis showed no startingmaterial. The reaction mixture was diluted with ethyl acetate, washedwith sat. NaHCO₃, water, saturated NaCl, dried (MgSO₄), and stripped invacuo. The residue was chromatographed on silica gel using 20%EtOAc/hexanes to give the product. ¹H NMR (300 MHz, CDCl₃): δ 8.91 (d,1H), 8.39 (m, 2H), 8.08 (m, 2H), 7.75 (m, 1H), 7.61 (m, 1H), 7.48 (m,2H), 7.46 (m, 1H), 6.87 (m 1H), 5.74 (s, 2H), 5.40-5.50 (m, 1H), 4.40(m, 1H), 3.60 (m, 2H), 3.25 (m, 1H), 3.07 (m, 1H), 2.27 (m, 2H),1.30-1.90 (m, 6H), 0.99 (m, 2H), 0.08 (s, 9H). LC/MS: 557 (M+H)⁺.

Step 2:3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Lithium hydroxide (22.7 mg, 0.000948 mol) was added to a solution of(1S,3S)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentylbenzoate and(1R,3R)-3-{2-cyano-1-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]ethyl}cyclopentylbenzoate (440 mg, 0.00079 mol) dissolved in a mixture of 1,4-dioxane(10.0 mL, 0.128 mol), methanol (4.0 mL, 0.099 mol), and water (4.0 mL,0.22 mol) in a round-bottom flask. The resulting mixture was stirred for20 hours at which time LCMS analysis showed no starting material. Thereaction mixture was extracted with ethyl acetate and the organicextracts were washed with sat. NaHCO₃, water, saturated NaCl, dried(MgSO₄), and stripped in vacuo. The residue was used in the nextreaction without further purification. LC/MS: 453 (M+H)⁺.

Step 3:3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt and3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt

The mixture of3-[(1S,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilewas deprotected under the same conditions described in Example 1, Step5. The mixture was separated using chiral LC and further purified by LCto give the isomers as the trifluoroacetate salts. Column: ChiralPak IA,4.6×250 mm, 5 micron particle. Mobile phase: 30% Ethanol in hexanes.Flow Rate: 0.8 ml/min-analytical; Column: ChiralPak IA, 20×250 mm, 5micron particle. MobilePhase: 30% Ethanol in hexanes. Flow rate: 12ml/min preparative).

Pk1 [Retention time: 16.98 minutes]: ¹H (500 MHz, DMSO-d₆): δ 8.11 (brs,1H), 8.07 (brs, 1H), 7.70 (s, 1H), 7.03 (d, 1H), 6.46 (m, 1H), 3.80 (m,1H), 3.43 (m, 1H), 2.20 (m, 2H), 2.08 (m, 1H), 1.29 (m, 1H), 1.20 (m,1H), 0.60-0.90 (m, 4H). LC/MS: 323 (M+H)⁺.

Pk2 [Retention time: 18.68 minutes]: ¹H (500 MHz, CD₃OD): δ 8.91 (s,1H), 8.087 (s, 1H), 8.51 (s, 1H), 7.84 (d, 1H), 7.28 (m, 1H), 4.60 (m,1H), 4.34 (m, 1H), 3.20 (m, 2H), 2.91 (m, 1H), 1.92 (m, 2H), 1.60 (m,3H), 1.35 (m, 1H). LC/MS: 323 (M+H)⁺.

Pk3 and Pk4 eluted together (23.13 minutes).

Example 33-[(1S)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetate salt and3-[(1R)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt

Step 1:3-[(1S)-3-oxocyclopentyl-3-[4-(7-[2-(trimethylsilyl)ethoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-]H-pyrazol-1-yl]propanenitrileand3-[(]R)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

Dimethyl sulfoxide (0.340 mL, 4.79 mmol) was added to a solution ofoxalyl chloride (0.20 mL, 2.4 mmol) in methylene chloride (25 mL) −78°C. in a round-bottom flask. The resulting mixture was stirred at −78° C.for 15 minutes and a solution of3-[(1S,3R)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R,3S)-3-hydroxycyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(0.84 g, 1.8 mmol) in methylene chloride (17 mL) was added dropwise. Theresulting mixture was stirred at −78° C. for 60 minutes andtriethylamine (0.722 mL, 5.18 mmol) was added. After stirring at −78° C.for 60 minutes, the reaction mixture was warmed to 0° C. and stirred for1 hour. The reaction mixture was diluted with ethyl acetate, washed withwater, saturated NaCl, dried (MgSO₄), and stripped in vacuo. The residuewas purified by chromatography on silica gel using 40% EtOAc/hexanes togive the product. ¹H NMR (300 MHz, CDCl₃): δ 8.91 (m, 1H), 8.40 (d, 1H),8.38 (s, 1H), 7.47 (m, 1H), 6.85 (t, 1H), 5.74 (s, 2H), 4.51 (m, 1H),3.60 (t, 2H), 3.00-3.30 (m, 3H), 1.50-2.70 (m, 6H), 0.98 (t, 2H), 0.00(s, 9H). LC/MS: 451 (M+H)⁺.

Step 2:3-[(1S)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetate salt and3-[(1R)-3-oxocyclopentyl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitriletrifluoroacetic acid salt

The mixture of3-[(1S)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand3-[(1R)-3-oxocyclopentyl]-3-[4-(7-[2-(trimethylsilyl)ethoxy]methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilewas deprotected under conditions similar to Example 1, Step 5, to givethe two diastereomeric ketones which were separated by chiralchromatography and purified by LC to give the diastereomers andenantiomers as the trifluoroacetate salts. Column: ChiralPak IA, 4.6×250mm, 5 micron particle. Mobile phase: 30% Ethanol in hexanes. Flow Rate:0.8 ml/min-analytical; Column: ChiralPak IA, 20×250 mm, 5 micronparticle. MobilePhase: 30% Ethanol in hexanes. Flow rate: 12 ml/minpreparative).

Pk1 [Retention time: 11.82 minutes].

Pk2 [Retention time: 13.94 minutes]: ¹H (500 MHz, CDCl₃): δ 10.07 (brs,1H), 8.79 (brs, 1H), 8.27 (s, 1H), 8.25 (s, 1H), 7.32 (d, 1H), 6.71 (m,1H), 4.40 (m, 1H), 3.12 (m, 1H), 2.97 (m, 2H), 2.00-2.32 (m, 5H), 1.61(m, 1H). LC/MS: 321 (M+H)⁺.

Pk3 [Retention time: 17.61 minutes]: ¹H (500 MHz, CDCl₃): δ 10.70 (brs,1H), 8.83 (brs, 1H), 8.34 (s, 1H), 8.30 (s, 1H), 7.35 (d, 1H), 6.73 (m,1H), 4.37 (m, 1H), 3.10 (m, 1H), 2.90 (m, 2H), 2.51 (m, 1H), 2.27 (m,1H), 2.15 (m, 1H), 1.91 (m, 1H), 1.84 (m, 1H), 1.60 (m, 1H). LC/MS: 321(M+H)⁺.

Pk4 [Retention time: 20.31 minutes].

Example A

TABLE 2 JAK 1 JAK 2 JAK 3 Fraction Human IC50 IC50 IC50 Unbound (%Intrinsic CL Compound (nM) (nM) (nM) human serum) (L/h/kg) Compound 1<10 <10 <10 <5 0.68 Metabolite 1 2.5-12 0.7-2.5 8.3-45  26-35 <0.50Metabolite 2   3-15   2-2.8 17-30  5-27 <0.50 Metabolite 3 2.7-122.1-5.9 11-41 14-56 <0.57

Metabolites 1, 2, and 3 were isolated from rat or dog urine afteradministration of Compound 1 in connection with pharmacokinetic andtoxicokinetic studies. Activity data for Metabolites 1, 2, and 3, alongwith free fraction and intrinsic clearance data, was compared with thatfor the parent compound, Compound 1. JAK activity assays, free fractionassays, and intrinsic clearance assays are described below. Data pointswere obtained for some individual stereoisomers of Metabolites 1, 2, and3, and the numerical range provided above reflects the highest andlowest values obtained for all the stereoisomers tested. As can be seenin Table 1, the metabolites are potent inhibitors of JAK1, JAK2, andJAK3, like Compound 1. However, the free fractions obtained for themetabolites are unexpectedly higher and the intrinsic clearancedesirably lower than for Compound 1.

In Vitro JAK Kinase Assay

Compounds herein were tested for inhibitory activity of JAK targetsaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a.781-1124) with an N-terminal His tag were expressed using baculovirus ininsect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3was assayed by measuring the phosphorylation of a biotinylated peptide.The phosphorylated peptide was detected by homogenous time resolvedfluorescence (HTRF). IC₅₀s of compounds were measured for each kinase inthe reactions that contain the enzyme, ATP and 500 nM peptide in 50 mMTris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%)BSA. The ATP concentration in the reactions was 90 μM for Jak1, 30 μMfor Jak2 and 3 μM for Jak3. Reactions were carried out at roomtemperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nMSA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.).Binding to the Europium labeled antibody took place for 40 minutes andHTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston,Mass.). Compounds having an IC₅₀ of 10 μM or less for any of theabove-mentioned JAK targets were considered active.

Free Fraction Assay

The protein binding of a test compound was determined by equilibriumdialysis using a Dianorm system from Harvard Apparatus (Holliston,Mass.). The dialysis was performed at 37° C. for 2 hrs in human serum.The metabolites were incubated at 3 μM, and Compound 1 at 3 and 10 μM.The compound concentrations in serum and buffer post-dialysis weredetermined by LC/MS/MS analysis. Free fraction is defined as the ratioof the buffer versus serum concentration.

Intrinsic Clearance Assay

Intrinsic clearance was determined by incubating 1 μM of test compoundin human mixed gender liver microsomes (0.5 mg/mL protein) at 37° C. inthe presence of 1 mM NADPH. The disappearance of the test compound wasmonitored by LC/MS at 0, 5, 10, 20 and 30 min. The slope of decline incompound concentration was used to calculate the human intrinsicclearance by employing standard methods reported in the literature.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

1-53. (canceled)
 54. A method of treating a myeloproliferative disorderin a patient in need thereof, comprising administering to the patient ametabolite selected from:3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile;3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile;and3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxycyclopentyl)propanenitrile,wherein the metabolite is generated as a result of administration of(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrileor a pharmaceutically acceptable salt thereof to the patient.
 55. Themethod of claim 54, wherein the myeloproliferative disorder ispolycythemia vera (PV).
 56. The method of claim 54, wherein themyeloproliferative disorder is essential thrombocythemia (ET).
 57. Themethod of claim 54, wherein the myeloproliferative disorder is myeloidmetaplasia with myelofibrosis (MMM).
 58. The method of claim 54, whereinthe metabolite is3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-hydroxycyclopentyl)propanenitrile.59. The method of claim 54, wherein the metabolite is3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(2-hydroxycyclopentyl)propanenitrile.60. The method of claim 54, wherein the metabolite is3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-(3-oxycyclopentyl)propanenitrile.